Passive immunization is an important tool to dissect the nature of protective immune responses. In the current funding period, we have provided proof-of-concept that combinations of human broadly reactive neutralizing monoclonal antibodies (brnmAbs) can completely protect neonatal rhesus monkeys from oral simian-human immunodeficiency virus (SHIV) infection, even as post-exposure prophylaxis. Our oral R5 SHIV challenge model has a dual function: 1) to determine protective epitopes using passive immunization with brnmAbs, and 2) to develop immunoprophylaxis for intrapartum HIV transmission and transmission via breast feeding. The Specific Aims for the Merit Extension period are to: 1. Generate biologically relevant, R5-tropic SHIV strains carrying clade A or D env genes to represent clades prevalent in areas of the world with high HIV infection rates (we have already established a mucosal SHIV-clade C transmission model). R5 SHIV-clade A and SHIV-clade D viruses will be adapted to rhesus monkeys by rapid serial passage. We will take advantage of the optimized proviral backbones we created for our SHIV-clade C, which was engineered to contain additional NF[unreadable]B sites in the proviral long terminal repeat (LTR) to boost viral replicative capacity in vivo. 2. Establish repeated low-dose oral challenge models in infant rhesus macaques with the R5 SHIV-clade A (or D) strains generated in Aim 1. 3. Isolate novel nmAbs through screening of Fab libraries obtained from bone marrow aspirates of monkeys with high-titer, cross-clade neutralizing antibody (nAb) responses, in collaboration with Dr. Dimiter Dimitrov. BrnmAbs will also be tested for anti-self reactivity. 4. Develop novel nmAb combinations for passive immunization in our neonatal primate/oral challenge model with R5 SHIVs. In Aim 4a, we will test whether novel brnmAbs can be combined to increase the potency of neutralization in vitro. In Aim 4b, we will evaluate such combinations in infant rhesus monkeys for safety and efficacy against oral challenge with R5 SHIV-clade A and SHIV-clade D viruses. Our experiments will not only contribute to important SHIV reagents for AIDS vaccine development, but also further understanding of neutralizing HIV-1 Env epitopes. As our track record shows, the neonatal monkey model has the proven capability of identifying protective nmAb epitopes. This information may be of vital importance for developing protective AIDS vaccines in the future. Lastly, if safety and efficacy profiles of our new nmAb combinations are acceptable, we may yet achieve our initial goal, the prevention of MTCT through passive immunization, possibly combined with active immunization in the future.